Herpes simplex virus type 2 (HSV-2) is the leading cause of genital herpes worldwide, the most common infection associated with neonatal encephalitis, and a major co-factor for HIV acquisition and transmission. No vaccine is currently available and recent clinical prevention trials with candidate vaginal microbicides have not been successful. These facts make it imperative to identify new targets and strategies for prevention. Development of new approaches requires an understanding of the cellular events critical for the establishment of primary and recurrent infection and for spread of virus from cell- to-cell. The studies proposed in this competitive renewal address this priority by defining how HSV-2 usurps host cell calcium signaling pathways to promote entry, facilitate cell- to-cell spread between epithelial cells and from neuronal to non-neuronal targets. Results of studies completed during the first funding period support a novel paradigm for viral entry in which calcium plays a pivotal role. In summary, we found that interactions between glycoprotein D (gD) and nectin-1 trigger an increase in calcium near the plasma membrane, which may be required to initiate viral entry. However, delivery of viral capsids intracellularly requires the release of intracellular endoplasmic reticulum (Ca2+ stores, which is only observed when glycoproteins(g) B, D, and the heterodimeric complex gH/gL are present and requires integrin and inositol (1,4,5)-triphosphate receptor (IP3R) signaling. Blockade of these signaling pathways prevents viral entry and impedes cell-to-cell spread. Notably, the Ca2+ response differs when cells express herpes virus entry mediator (HVEM), rather than nectin-1, as their primary gD receptor. Moreover, when experiments are conducted with polarized cells, an experimental model that more closely replicates what happens clinically, differences in Ca2+ signaling are observed at the apical and basolateral surfaces. HSV preferentially infects the apical membrane with rapid release of intracellular Ca2+, whereas entry from the basolateral surface is less efficient and is associated with a delayed Ca2+ response. These observations are consistent with apical sorting of receptors involved in the calcium signaling response. Building from this foundation, we will further define the role Ca2+ signaling plays in HSV entry and extend this work to uncover the signaling pathways critical for HSV spread between epithelial cells and keratinocytes and from neuronal to non-neuronal cells with the overarching goal of identifying novel targets for prevention. Genetic, biochemical, and cell biological approaches will be used that will include targeted protein silencing with small interfering RNA, use of pharmacological antagonists and agonists;and fluorescence-based confocal laser microscopy approaches. This investigation will provide new opportunities for development of novel antiviral strategies to prevent primary and recurrent infection.